FNDC5/Irisin attenuates diabetic cardiomyopathy in a type 2 diabetes mouse model by activation of integrin αV/β5-AKT signaling and reduction of oxidative/nitrosative stress

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Highlights

  • Cardiac FNDC5/irisin and plasma irisin were downregulated in diabetic db/db mice compared to the db/+ controls.

  • Exogenous FNDC5/irisin therapy attenuated diastolic dysfunction and cardiac remodeling in db/db mice.

  • Suppression of mitochondria-dependent apoptosis may be responsible for the cardioprotection of FNDC5/irisin

  • Activation of integrin αV/β5-AKT signaling was responsible for the cardioprotective effects of FNDC5/irisin.

Abstract

Irisin, the cleaved form of the fibronectin type III domain containing 5 (FNDC5) protein, is involved in metabolism and inflammation. Recent findings indicated that irisin participated in cardiovascular physiology and pathology. In this study, we investigated the effects of FNDC5/irisin on diabetic cardiomyopathy (DCM) in type 2 diabetic db/db mice. Downregulation of myocardial FNDC5/irisin protein expression and plasma irisin levels was observed in db/db mice compared to db/+ controls. Moreover, echocardiography revealed that db/db mice exhibited normal cardiac systolic function and impaired diastolic function. Adverse structural remodeling, including cardiomyocyte apoptosis, myocardial fibrosis, and cardiac hypertrophy were observed in the hearts of db/db mice. Sixteen-week-old db/db mice were intramyocardially injected with adenovirus encoding FNDC5 or treated with recombinant human irisin via a peritoneal implant osmotic pump for 4 weeks. Both overexpression of myocardial FNDC5 and exogenous irisin administration attenuated diastolic dysfunction and cardiac structural remodeling in db/db mice. Results from in vitro studies revealed that FNDC5/irisin protein expression was decreased in high glucose (HG)/high fat (HF)-treated cardiomyocytes. Increased levels of inducible nitric oxide synthase (iNOS), NADPH oxidase 2 (NOX2), 3-nitrotyrosine (3-NT), reactive oxygen species (ROS), and peroxynitrite (ONOO) in HG/HF-treated H9C2 cells provided evidence of oxidative/nitrosative stress, which was alleviated by treatment with FNDC5/irisin. Moreover, the mitochondria membrane potential (ΔΨm) was decreased and cytochrome C was released from mitochondria with increased levels of cleaved caspase-3 in HG/HF-treated H9C2 cells, indicating the presence of mitochondria-dependent apoptosis, which was partially reversed by FNDC5/irisin treatment. Mechanistic studies showed that activation of integrin αVβ5-AKT signaling and attenuation of oxidative/nitrosative stress were responsible for the cardioprotective effects of FNDC5/irisin. Therefore, FNDC5/irisin mediates cardioprotection in DCM by inhibiting myocardial apoptosis, myocardial fibrosis, and cardiac hypertrophy. These findings implicate that FNDC5/irisin as a potential therapeutic intervention for DCM, especially in type 2 diabetes mellitus (T2DM).

Graphical abstract

T2DM results in a decrease in protein expression of FNDC5/irisin and plasma levels of irisin. Myocardial FNDC5 overexpression or irisin supplementation increases circulating irisin. Irisin binds to integrin αV/β5 receptor and activates AKT, which then reduces iNOS/NOX2, attenuates oxidative/nitrosative stress, and inhibits mitochondria-dependent apoptosis. The inhibition of cardiomyocyte apoptosis by FNDC5/irisin contributes to the attenuation of cardiac hypertrophy, fibrosis, and diastolic dysfunction in db/db mice with overexpression of myocardial FNDC5 or by irisin supplementation.

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Introduction

Worldwide, the incidence and prevalence of diabetes mellitus (DM) are increasing rapidly. It is estimated that by 2030, over 578 million people worldwide will be diagnosed with DM [1]. Type 2 diabetes mellitus (T2DM) accounts for 90%–95% of all cases of diabetes [2]. Patients with T2DM have a considerably higher risk of cardiovascular morbidity and mortality [3]. Diabetic cardiomyopathy (DCM), first reported by Rubler et al. in 1972 [4], is a specific type of cardiomyopathy found in patients with DM. The condition is manifested in these patients as symptoms of heart failure in the absence of coronary artery disease, hypertension, or valvular heart disease. DCM is an independent diabetic cardiac complication that can cause pathologic abnormalities including cardiomyocyte apoptosis, left ventricular dysfunction, cardiac remodeling, inflammation, oxidative/nitrosative stress, and myocardial metabolic disturbance [5]. In the early stages, DCM is characterized by cardiomyocyte apoptosis, myocardial fibrosis, and diastolic heart failure with preserved ejection fraction (HFpEF). In the later stage, heart failure with reduced ejection fraction (HFrEF) becomes evident [6]. Inhibition of the early cardiac apoptosis can prevent subsequent DCM [7,8]. The outcomes of current therapeutic strategies for preventing DCM are far from satisfactory. Therefore, novel pharmacological or molecular interventions to curb the escalating incidence of ventricular dysfunction and remodeling in DCM patients are of utmost importance.

Diabetes is an independent risk factor for heart failure [9], which can be divided into two distinct entities: HFrEF and HFpEF. HFrEF is associated with systolic dysfunction, characterized by the inability of the myocardium to contract and eject enough blood. HFpEF is linked to diastolic dysfunction, whereas systolic function remains normal or near normal (preserved EF). HFpEF patients are generally older and can have numerous comorbidities, including hypertension, DM, chronic kidney disease, and obesity [10]. Importantly, about 27–45% of patients with HFpEF have DM [10,11]. In diabetic patients, abnormal diastolic filling is an early sign of left ventricular (LV) dysfunction [11]. Worldwide, HFpEF is a growing epidemiological clinical problem, with a prevalence that has markedly grown due to the increasing prevalence of risk factors in an aging population [12]. In general, HFpEF precedes HFrEF, which highlights the importance of early detection and timely intervention of diastolic dysfunction in T2DM patients. Leptin receptor knockout mice, also known as db/db mice, are commonly used as a mouse model that recapitulates the diastolic dysfunction and HFpEF observed in diabetic patients [13,14].

Irisin is a novel myokine that is secreted mainly by myocytes, including skeletal and heart tissue [15]. Irisin is a cleaved protein derived from the full-length fibronectin type III domain containing 5 (FNDC5) [16]. It has been reported that irisin mediates the beneficial effects of exercise on the browning of adipocytes and thermogenesis by stimulating uncoupling protein 1 expression [17]. Apart from its beneficial role in metabolic disorders, numerous studies have indicated the protective effects of irisin on cardiovascular diseases, including atherosclerosis, myocardial infarction, and transverse aortic constriction-induced cardiac hypertrophy [[18], [19], [20], [21]]. However, the role of FNDC5/irisin in DCM induced specifically by T2DM remains largely undetermined. The aims of the current study were as follows: 1) investigate the potential of myocardial FNDC5 overexpression or irisin administration to protect the heart from adverse changes in T2DM; and 2) clarify the underlying mechanisms of action involved.

Section snippets

Experimental animals

All animal experiments were performed according to the National Institutes of Health guidelines for the use of laboratory animals (NIH publication No. 85–23, revised 2011) and were approved by the Fourth Military Medical University Animal Use and Care Committee. Adult male leptin receptor-deficient (db/db) mice and their non-diabetic littermates (db/+) mice were purchased from Changzhou Cavens Laboratory Animal Co. Ltd. (Jiangsu, China). Mice were housed under standard laboratory conditions and

FNDC5/irisin was downregulated in db/db hearts or HG/HF-treated cardiomyocytes

The patterns of FNDC5/irisin expression in diabetes remain to be clarified. We previously reported that serum levels of irisin were significantly reduced in T2DM mice compared with the levels in non-diabetic mice [26,27]. In the present study, protein levels of FNDC5 and irisin were lower in the hearts of db/db mice compared to those in lean db/+ controls (Fig. 1A). Interestingly, treatment with HG/HF significantly reduced protein expression of FNDC5 and irisin in primary NRVMs (Online Fig. 1,

Discussion

DCM initially manifests as isolated diastolic dysfunction and progresses to systolic dysfunction over time. Although the etiologies of type 1 diabetes mellitus (T1DM) and T2DM differ, defects in systemic metabolism including hyperglycemia and dyslipidemia occur in both diseases. During the early stages of diabetes, a lack of insulin or insulin resistance induces a metabolic shift in cardiomyocytes, resulting in an increase in the uptake of free fatty acids by cardiomyocytes. The excessive

Conclusions

We found that myocardial FNDC5/irisin and plasma irisin are downregulated in T2DM mice, and that supplementation of FNDC5/irisin protects against cardiac diastolic dysfunction by attenuation of cardiomyocyte apoptosis, cardiac hypertrophy, and cardiac fibrosis. Downregulation of oxidative/nitrosative stress and mitochondria-dependent apoptosis are the possible mechanisms involved in this process. As FNDC5 overexpression restored plasma irisin levels in db/db mice and increased the protein

Declaration of Competing Interest

None.

Acknowledgments

This work was financially supported by the National Key R & D Plan (Grant No. 2018YFA0107400), Program for National Science Funds of China (Grants No. 81970212, 82022004, 81730011, 81970721, 81900241, and 82000265), and Program for China Post-Doctoral Science Foundation Grant (No. 2019M663987).

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    Chen Lin, Yongzhen Guo, and Yunlong Xia contributed equally to this work.

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